The objective of this proposal is to evaluate the use of a newly discovered, highly conserved outer-membrane protein antigen of Neisseria gonorrhoeae, designated NspA, as a potential candidate vaccine against gonorrhea, when administered by mucosal routes designed to induce high levels of antibodies in the genital tract. This will be accomplished by exploiting a novel technology, developed in this laboratory, for fusing bacterial protein antigens to the A2 subunit of cholera toxin (CT) and co-expressing the fusion protein with the nontoxic binding (B) subunit of CT, to form chimeric immunogens of the form NspA-CTA2/B, in which the toxic A1 subunit of CT has been replaced by the desired antigen. Chemical conjugates of NspA and CTB will also be evaluated. Alternative constructs will utilize type II heat-labile enterotoxins of Escherichia coli, which have different binding properties. Immunogens of this type have previously been shown to induce strong mucosa and circulating antibody responses when administered by mucosal routes. Specific IgA and IgG antibody responses in the genital tract (and other mucosal sites) and in the serum will be determined in mice immunized with these constructs as applied by intranasal or intragastric routes. Specific antibody secreting cells, specific T cells and the cytokines secreted by T cells will also be evaluated to assess the immune response in detail. A newly described mouse model of genital tract colonization by N. gonorrhoeae will be used to determine the ability of NspA-CTA2/B chimeric proteins and other constructs to elicit protective immunity against gonococcal infection. Potential mechanisms by which the expected IgA and IgG antibodies to NspA may be effective in protection against gonococcal infection of the genital tract will be examined by developing monoclonal IgA and IgG antibodies from mice mucosally immunized with NspA-CTA2/B constructs, and testing their ability to inhibit gonococcal adherence to and invasion of epithelial cells in culture, and to suppress genital colonization of mice with N. gonorrhoeae. The successful accomplishment of these objectives should provide a basis for further considering NspA as a component of a vaccine against gonorrhea, and for proposing trials designed to evaluate human genital tract immune responses to NspA-CTA2/B chimeric proteins. The information gained about genital tract immunity and the techniques used may also be applicable to other sexually transmitted diseases.